1. Field of the Invention
This invention relates to a composition, method and test kit. More specifically, this invention is directed to a molybdenum coordination complex, a method for use of such complex in analysis of biological fluids and a test kit for performance of such analysis. This invention is suitable for use in enhancement in accuracy of methods for immunochemical analysis of biological samples obtained from individuals (e.g. newborn infants and nursing infants of drug abusers) that have been inadvertently exposed to substances that can detract from detection of analytes of interest, specifically analytes that are endogenous to biological fluids which are the object of immunochemical analysis.
2. Description of the Prior Art
The diagnostic testing of biological fluids by clinical chemistry and immunochemical analysis is a well established a tool for confirmation of wellness or detection of a disease state. Notwithstanding the wide spread use and acceptance of such tests, the uniqueness of each fluid sample that is subjected to analysis, can introduce potential error into such assays. For example, a lipemic sample can interfere with clinical chemistry analysis by spectrophotometric techniques because of energy losses associated with light scattering from the dissolve fat globules in such samples. Similarly, in immunochemical analysis, the presence of both materials that are endogenous and exogenous to the sample can interfere with either the detection of the analyte of interest, or produce indiscriminate interactions with the test kit reagents and, thus, a false positive or a false negative. These problems have not gone unappreciated either by clinicians or by companies that provide such clinical diagnostic assays.
In virtually all screening assays, the test sample (e.g., blood, urine, saliva, etc.) typically contains some waste materials and cellular matter, along with the analytes of interest. Depending upon the assay format of choice, some isolation (physically and/or optically) of the analyte of interest is required to permit its detection and/or its quantification. The screening assay used in the analysis for such heterogeneous biological fluids generally fall into one of three assay formats/protocols: (a) homogenous assay; (b) heterogeneous assay; and (c) solid phase assay (the latter being further distinguishable as either “immunochromatographic” and “radial partition”).
Homogeneous Test Format—In the homogenous assay format, (also commonly referred by the EMIT® trademark for the corresponding commercial product), a sample suspected of containing the analyte of interest, and an analyte mimic conjugated to an enzyme, are admixed, under binding conditions, with a solution containing an antibody specific for binding to an epitope on each of the analyte and analyte mimic/enzyme conjugate. Each of the analyte and analyte mimic/enzyme conjugate compete for available binding sites on the analyte specific antibody. After a suitable incubation period, each of the analyte and analyte mimic/enzyme conjugate arrive at an equilibrium relative to their binding and displacement vis-à-vis the antibody, and, at that juncture, an enzyme specific substrate is added to the mix. The amount of unbound analyte mimic/enzyme conjugate that remains free from association with the antibody retains it enzymatic activity and converts the substrate to a detectable indicator that is measured by standard spectrophotometric analysis. The indicator specifies can be either a chomophore or a flourophore. The relative concentration of indicator detected by such analysis is compared with a standard curve that correlates with the amount of analyte in the test sample.
The foregoing homogeneous test format has been described in U.S. Pat. No. 4,190,496, (issued Feb. 26, 1980, to Kenneth Rubenstein, et al., and assigned to Syva Company), which is herein incorporated by reference in its entirety. Insofar as the performance of such homogenous assay necessarily contemplates the interaction of test kit reagents and sample, and the subsequent measurement of test results within the same fluid environment, the potential for interference from other sample constituents and/or from unreacted test kit reagents, can produce a false or inaccurate results.
For example, in a homogenous assay specific for detection of ethyl alcohol using alcohol dehydrogenase as the reporter enzyme, endogenous dehydrogenase must be initially inhibited with an antibody specific for such endogenous dehydrogenase prior to performance of a homogenous assay for the analyte of interest, See U.S. Pat. No. 5,861,269, Methods For Removing Interferences Due To Endogenous Dehydrogenase In Enzyme Assays (issued Jan. 19, 1999, to McCormack et. al., and assigned to Dade Behring Marburg GmbH). There are also a number other reported instances involving comparable interference, depending upon the analyte and test kit reagent combinations used in such homogenous immunoassays.
Similarly, the use of enzyme specific antibodies have also been reported as necessary to modulate enzyme activity within such homogenous test environments to more accurately correlate such activity with the concentration of the analyte of interest in the test sample, See U.S. Pat. No. 5,972,630, Homogenous Immunoassay Using Enzyme Inhibitors, to Cromer et al. (issued Oct. 26, 1999, and assigned to Dade Behring Marburg GmbH). In the Cromer patent, an enzyme specific antibody and an enzyme labeled conjugate are added to the test sample concurrently. Each of the enzyme specific antibody and enzyme labeled conjugate are capable of binding to the analyte of interest and the enzyme activity of the test medium monitored in the traditional manner.
Solid Phase Test Format—Unlike the EMIT™ type of homogenous assay described above, solid phase enzyme immunoassays effect a physical separation of bound and unbound materials within a test site prior to addition of an enzyme specific substrate for the enzyme labeled conjugate that is bound to such test site. Representative solid phase diagnostic tests, based upon a radial partitioning chromatographic test format, is disclosed in U.S. Pat. No. 4,517,288 (to Giegel et al). In the latter system, the test sample is applied to immobilized binding material within a delimited area of a bibulous membrane. An excess of substrate solution is thereafter applied to this same area, thereby washing (radially displacing) the unbound materials from this delimited area into the surrounding area of the membrane. An indicator molecule is cleaved from the substrate by the immobilized enzyme, is monitored over a defined period of time, and the rate of formation of the indicator compared to a standard curve, which comparison indicates the concentration of analyte in the test sample.
In order to avoid some of the potential problems associated with the use of an enzyme labeled conjugate (e.g. incomplete separation of bound and free conjugate within the test site), a number of solid phase assays have been developed wherein the conjugate is labeled with colloidal gold or other comparable pigment or dye. Thus, as the conjugate is bound to the test site, its concentration at the test site gradually increase, as does the concentration of the colloidal gold, until it becomes visually detectable without further interaction with another test kit reagent (e.g. substrate). Representative solid phase diagnostic tests, based upon a linear chromatographic test format, are disclosed in U.S. Pat. No. 4,703,017 (to Campbell); U.S. Pat. No. 5,591,645 (to Rothstein); U.S. Pat. No. 5,073,484 (to Swanson); U.S. Pat. No. 5,602,040 (to May)—all of which are herein incorporated by reference in their entirety. Notwithstanding, the elimination of a reagent substrate does not otherwise reduce the possible interference from exogenous substances; and, the same physical and immunochemical constraints and limitations apply.
Irrespective of the indicator system, such solid phase immunoassays are not, however, free from interference by either endogenous or exogenous materials that may be present in the test sample. As discussed herein relative to homogenous assays, interferants can compete with test kit reagents to product false positive and thereby result in an elevated or false positive result; or, otherwise inhibit test kit reagents from interaction with the analyte of interest, producing a false negative result. To the extent that these interferants are present, their presence may be manifest by inhibition of the physical separation of bound and free materials within the solid phase, thus, precluding sufficient differentiation of the bound from the free fraction. Where this is inadequate separation of free from unbound materials within the solid phase, it is not possible to effectively measure the presence or amount of analyte at the test site.
The foregoing analytical testing of diagnostic fluids can also be complicated or frustrated by the presence of toxins and/or chemical substances (also “exogenous materials”) that have been inadvertently absorbed or ingested by the individual. These toxins or chemical substance typically are converted upon absorption by the body's metabolic processes and, thus, the presence of such toxins or chemical substance is generally manifest by analysis for metabolites of such toxins or chemical substances (also “metabolites” or “exogenous analytes”).
Long Felt Need In Prior Art—The exposure of newborn infants in the womb and, thereafter, during nursing, to toxins and chemical agents that are present in its birth mother's circulatory system and breast milk, is an all too common occurrence. Similarly, older individuals are also inadvertently exposed to similar toxins and chemical agents in the ordinary course of going about one's day-to-day life.
In newborns, and nursing infants born to drug abusers, such children often test positive for the drugs that are present within the circulatory system of the birth mother. The presence of such drugs, and the corresponding metabolites of such drugs in the biological fluids of such children, can often interfere with routine medical screening of such children for analytes that are monitored to confirm a wellness state or an abnormal condition associated with childhood development. Similarly, such toxins and chemical agents can be inadvertently inhaled, absorbed through the skin by an adult, or through physical contact with such toxins and agents and/or consumed orally incident to sharing a drink or dinner ware utensils. Moreover, the individual experiencing such exposure is often unaware such event or contact. This is particularly common in social settings, and in contacts with individuals that are relative strangers to one another. For example, it is not uncommon to attend a concert or a night spot and be exposed to second hand smoke from a controlled substances, such as marijuana and/or cocaine; or, to share a glass or a cigarette with a stranger in such social settings that may have been similarly contaminated with a controlled substance.
The amount or duration of an individual's contact with such toxins or agents need not be extensive for absorption to occur. Moreover, depending upon the circumstances surrounding such contact, such transient exposure can result in the appearance of the toxin or agent and/or metabolites thereof in the biological fluids of the person having undergone such inadvertent exposure. Notwithstanding that such exposure is brief, and the effect thereof is not overtly manifest, the presence of compounds indicative of such toxins and agents within the biological fluids of an individual, can have both medical, social and legal consequences.
For example, the limited and inadvertent exposure to relatively small amounts of second-hand smoke from a marijuana cigarette can result in metabolites (cannabinoids) remaining in tissues within the body for an extended period of time, and because of the sensitivity of current clinical chemistry analysis and immunodiagnostics, the presence of such compounds can be manifest in the analysis of biological fluids from such individuals for up to thirty (30) days following such exposure.
Typically, an analysis of biological fluids for endogenous analytes of interest in children and adults can be performed upon anyone of a number of different biological specimens and, in certain instances, upon extracts of samples of hair from such individuals undergoing such testing. The biological fluids of choice for performance of a screening analysis are generally obtained by non-invasive techniques; and, such fluids can include urine, saliva and/or a transudate (an interstitial fluid extracted through the skin). Where hair is the “sample” of choice, the living root from the hair strand is extracted and the extract thereof subjected to analysis. Alternatively, invasive methods can also be used to obtain a biological fluid (e.g. blood) for analysis. The venous and capillary blood samples obtained for this purpose are initially separated into their various fractions (cellular and plasma), and the plasma fraction subjected to further processing/dilution and analysis.
Heterogeneous Biological Fluid Samples—In each instance the sampling technique, as described herein, is indiscriminate as to its ability to confine the content of the sample to only those endogenous analytes of interest, or to the exclusion of compounds that can interfere with accurate detection and measurement of the endogenous analytes of interest. Thus, in the analysis of biological fluid samples and extracts from human tissues for analytes that are endogenous to body, the immunoreagents used therein can also interact compounds (metabolites) indicative of toxins and chemical agents that may have been inadvertently inhaled, absorbed through contact with the skin or ingested by the individual subjected to such testing. Where the biological fluid of choice is a waste material (e.g. urine, feces, etc.) produced by the individual's body, the presence of such toxins, chemical agents and/or their corresponding metabolite, (also “exogenous materials”), is most likely to occur. Conversely, where the fluid is an excretion from an organ or gland (e.g. saliva) the likelihood of interference of from an exogenous material is reduced. The analysis of venous and/or capillary blood tends to suffer from the same type of contamination as samples of human waste.
Where metabolites and/or compounds indicative of such toxins and chemical agents are present in the test sample of the biological fluid sample or tissue extract, such compounds may, in the subsequent analysis of such test sample, either (a) mask the presence of one or more of the endogenous analytes of interest by interacting with an analyte of interest, and thereby produce a false negative; (b) interfere with test kit reagents used to detect the endogenous analytes of interest and thereby produce a false negative; and/or (c) mimic the endogenous analyte of interest and thereby produce a false positive or an artificially elevated level of the analyte above the basal level for the analyte of interest within the sample. In each instance, the test result obtained does not accurately reflect the status of an individual's disease or wellness state, and can result in misdiagnosis of the individual that is undergoing such testing. In addition to the obvious medical consequences of an erroneous test results for such endogenous analyte of interest, such test may also manifest the presence of the compound indicative of exposure to a toxin or chemical substance, and, under certain circumstance, a produce a positive drug test.
In social terms, the positive testing for drugs of abuse from inadvertent exposure to second hand smoke can be devastating professionally, and, in addition, result in social/legal consequences that may never be resolved or purged from an individual's employment or criminal record.
As is evident from the foregoing discussion, there continues to exist a need to selectively isolate endogenous analytes within a test sample from toxins or chemicals, or metabolites of toxins or chemicals, that can potentially interfere with detection of such endogenous analytes. In order to be effective, the materials and methods used in such isolation cannot otherwise interfere with either the test kit chemistries or the test protocol or, in automated systems, the performance of the test instrument (e.g. test cycle time, reading of a reagent blank, reading of sample optical interferants, etc.), used in the detection of the endogenous analytes of interest.